MicroRNA-4516 suppresses proliferative vitreoretinopathy development via negatively regulating OTX1

Proliferative vitreoretinopathy (PVR) progression is associated with TGF-β2-induced epithelial–mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. In cancer cells, miR-4516 downregulates orthodenticle homeobox 1 (OTX1)-mediated cell invasion. Moreover, OTX1 is shown to be involved in invasion and EMT. The purpose of this study was to assess whether microRNA (miR-4516) suppresses EMT in RPE cells. EMT features were assessed using Western blotting, immunocytochemical staining, scratch-wound healing, modified Boyden chamber assay, and collagen gel contraction assay. For in vivo testing, a rabbit model was used, which involved induction of PVR by injection of transfected spontaneously arising RPE (ARPE) cells into the vitreous chamber. The putative target of miR-4516 was identified by luciferase reporter assay. Results showed that TGF-β2-induced transdifferentiation and migration of RPE cells was inhibited by miR-4516 delivery. Overexpression of miR-4516 led to upregulation of zonula occludens-1, downregulation of α-smooth muscle actin and vimentin, and cell contractility—all EMT features—in the TGF-β2-treated ARPE-19 cells. MiR-4516 regulated OTX1 expression negatively by binding to its 3’-UTR. TGF-β2-induced phosphorylated ERK was inhibited in miR-4516-overexpressing ARPE-19 cells. MiR-4516 suppressed experimental PVR in vitro and in vivo. In conclusion, the overexpression of miR-4516 suppresses TGF-β2-induced EMT in a PVR model, and its role in PVR depends on OTX1/ERK. Further research is needed to develop a feasible treatment method to prevent and treat PVR.


Introduction
The main pathogenesis of proliferative vitreous retinopathy (PVR) involves epithelial-mesenchymal transformation (EMT) of retinal pigment epithelium (RPE) cells due to disorderly growth factors (e.g. TGF-β) in vitreous humor [1][2][3][4]. EMT results in a morphological transition of epithelial cells to a mesenchymal phenotype characterized by (i) reassembly of the

Cell cultures and treatment
The human RPE cell line ARPE-19 was obtained from the American Type Culture Collection (Manassas, VA, USA). ARPE-19 cells were cultured in a 1:1 mixture of Dulbecco's Modified Eagle Medium (DMEM) and nutrient mixture F-12 Ham (Invitrogen-Gibco, Grand Island, NY, USA), which contained 4 mM L-glutamine, to which 10% fetal bovine serum (FBS; Invitrogen-Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich, St Louis, MO, USA) were added. The cells were kept in a humidified environment of 5% CO2 at 37˚C, and the medium was exchanged twice a week. ARPE-19 cells were transfected with miR-4516 or miR-negative control (miR-NC; Life Technologies, Carlsbad, CA, USA) for 24 hours, and then with or without 10 ng/mL human recombinant TGF-β2 (PeproTech, New York, NY, USA) for 24 hours extra. Unless otherwise stated, all experiments were performed under serum-free conditions.

Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR)
After removing the medium and rinsing with phosphate buffered saline (PBS) 3 times, total RNA was extracted from the cells using TRIzol reagent (Life Technologies). Next, reverse transcription of an aliquot of 1μgof total RNA using TaqMan MicroRNA Reverse Transcription Kit. Real MOD Probe SF 2X qPCR mix and Step One Plus PCR System (Applied Biosystems, Foster City, CA, USA) were used for quantitative real-time PCR (RT-qPCR). The 2 -ΔΔCT method was used to normalize the expression level of miR-4516 to the expression level of control (U6 small nuclear RNA) or miR-361. The expression level of miR-4516 in the presence or absence of 10ng/mL TGF-β2 in RPE cells was also evaluated.

Western blotting
After the experimental treatments, the cells were lysed and sonicated in lysis buffer (50 mMTris-HCl [pH 7.5], 2% sodium dodecyl sulfate, and 1 mM phenylmethylsulfonyl fluoride). The protein content was quantified using the Pierce BCA protein assay kit (Rockford, IL, USA). Specifically, 20 μg of protein in each sample was loaded and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). After blocking with 5% skimmed milk dissolved in a phosphate buffer solution with Tween20 at room temperature for 1 hour, the membranes were directly probed with primary antibodies against α-smooth muscle actin (α-SMA; Sigma-Aldrich, St. Louis, MO, USA), anti-zonula occludens (ZO)-1 (Zymed Laboratories, South San Francisco, CA, USA), phosphorylated (p)-ERK1/2 (1:1000 dilution), ERK1/2 (1:1000 dilution), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:25,000dilution) overnight at 4˚C and then incubated with the horseradish peroxidase-conjugated secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA, USA) for 1 hour. Next, the proteins were visualized using enhanced chemiluminescence (ECL; Millipore). The Western blotting images were acquired using the UVP BioSpectrum 500 imaging system (Wolflabs Ltd., Pocklington, England) and examined using Vision Works LS Analysis software (Upland, CA, USA). The expression level of GAPDH was used as a loading control.

Immunocytochemistry
The ARPE-19 cells were seeded into 8-well cell chamber slides and transfected with miR-4516 or miR-NC. Next, they were incubated for up to 24 h in the presence or absence of TGF-β2.

Scratch wound healing assay
A modified in vitro scratch test was used to assess cell migration in vitro. In short, 95% confluent ARPE-19 cells were serum starved for 24 hours and pretreated with 10 μg mitomycin-C for 2 hours to suppress cell proliferation. A sterile 200 μL pipette tip was then used to inflict scratches on the monolayer. Next, the medium was removed and replaced with fresh serumfree medium containing the test substance (TGF-β2, miR-4516, or miR-NC) or control. The Olympus IX70 microscope equipped with a digital camera was used to photograph selected areas at 4x magnifications of 24 and 48 caps. The width of the scratch was measured using Image J software to calculate the distance between its two edges. The wound healing at the central observation area were quantified at 24 and 48 hours by Image J software.

Cell migration assay
The modified Boden chamber assay was also used to assess cell migration. In short, ARPE-19 cells were seeded in the upper chamber of a 24-well plate coated with fibronectin at a density of 5×10 4 cells/well (Corning Inc., Corning, NY, USA) with a pore size of 8 μm (ARPE-19 cells could pass this pore size). The lower chamber was inoculated with 0.1% FBS-DMEM-F12 containing 10 ng/mL TGF-β2. After 5 hours of incubation, the cells that migrated to the lower side (insert) were washed with PBS, fixed with cold methanol (4˚C) for 10 minutes, and then counterstained with crystal violet (Thermo Fischer Scientific) for 20 minutes. The numbers of migrated cells were counted using a phase contrast microscope. Each insert counts four randomly selected fields.

Collagen gel contraction assay
Some modifications were made to evaluate collagen gel shrinkage. In short, rat tail type I collagen (Sigma-Aldrich) was dissolved in 0.1% acetic acid in sterile distilled water and stored at 4˚C overnight. The 24-well plate was coated with 2% FBS overnight to block non-specific binding. ARPE-19 cells (1.0×10 6 cells/mL) were resuspended in serum-free DMEM-F12. The cell suspension was mixed with 5.0 mL of 3 mg/mL type I collagen, 3.0 mL of concentrated serum-free DMEM-F12 containing glutamine and antibiotics, and 391 μL of 1 mM NaOH. Next, 350 μL of the mixture was added to each FBS-coated well and solidified by incubating in 5% CO2 at 37˚C for 1 hour. After 1.5 hours, the collagen gel was removed from the bottom. A micro spatula was used to add 1 mL of 10% FBS-DMEM-F12 to the top of each gel. After 24 hours, the medium was removed, the gel was washed with serum-free DMEM-F12, and incubated in serum-free DMEM-F12 containing 10ng/mL TGF-β2 at 37˚C for another 3 days. The cell culture medium was changed every other day. Collagen gel without RPE cells was used as baseline data for contraction. On the third day, an LAS-3000 Charge Coupled Device Camera (Fujifilm, Dusseldorf, Germany) was used to observe, record and measure the surface area of each matrix. The percentage of gel shrinkage was calculated as follows: [(gel size on day 1-gel size on day 3)/gel size on day 1] × 100. All experiments should be conducted at least 3 times.

Luciferase reporter assay
The putative binding site of miR-4516 in the 3'-UTR of OTX1 or complementary DNA fragments and the mutant 3'-UTR of OTX1 were amplified and subcloned into the pGL3 luciferase reporter vector (Promega, Madison, Wisconsin, USA). The cells were seeded into a 6-well plate at a density of 1 × 10 5 cells/well, and then co-transfected with vector (original or mutant 3'-UTR containing OTX1) and miR-4516. After 24 hours of incubation, the cells were collected and prepared with reporter lysis buffer. Next, the dual luciferase reporter gene detection system (Promega Corp, Madison, WI, USA) was used to measure the luciferase activity in each well.

In vivo inhibition of PVR progression by miR-4516 in an animal model
Five New Zealand albino rabbits, each weighing 1.5 kg, were used for the animal model. Basic animal research was approved and performed by the Institutional Animal Care and Use Committee of Taipei National Defense Medical Center, Taiwan (recognized by the International Association for Laboratory Animal Care Evaluation and Certification) (No: IACUC-21-229). The rabbits received humane care as outlined in the "Guidelines for the Care and Use of Laboratory Animals." The animals were kept under standard conditions (20±11˚C, 12 hours light/ 12 hours dark cycle), with a floor area of 1.5 square feet and a height of 16 inches. A sufficient amount of food and fresh water equipment was provided to accommodate the rabbits' visits at any time. During the experiment, rabbits were observed daily and checked for activities. All animal experiments were conducted in accordance with the Vision and Ophthalmology Research Association statement on the use of animals in ophthalmology and vision research. After dilation of the pupils by instillation of eye drops containing 5% phenylephrine and 1% tropicamide (2 drops per drop), the rabbits were anesthetized with 50 mg/kg Zoletil and 10 mg/kg xylazine. A 31-guage needle was passed through the sclera 2.5 mm behind the scleral limbus, and 0.1 mL of 10 ng/mL TGF-β2 and 0.1 mL of balanced salt solution (BSS) containing approximately 1 × 10 6 ARPE-19 cells transfected with miR-4516 or miR-NC was injected directly over the optic disc of the right eye. On days 1, 8, 15, and 22, a total of four doses were administered to each eye.
After the injections were administered, the rabbits underwent indirect ophthalmoscope examination twice a week for 5 weeks (days 1-37). PVR progress was determined using the Fastenberg classification. The staging criteria are listed in S1 Table. After the experiment, the rabbits were euthanized in a CO 2 chamber. After exposure to CO 2 , each rabbit was placed in room temperature air for 20 minutes to allow for possible recovery.

H&E staining
All rabbit eyes were re-fixed in neutral buffered formalin, embedded in paraffin, and sectioned (5μm thick). Serial sections closest to the defect center were then collected, floated in a 40˚C water bath, placed on organosilane-coated (silylated) microscope slides, and baked overnight at 37˚C. The sections were de-paraffinized in xylene and rehydrated in continuous ethanol rinses for hematoxylin and eosin staining.

Statistical analysis
Data are expressed as mean ± standard error of mean. All experiments were performed in triplicate and repeated at least three times. GraphPad Prism for Windows version 5.01 (GraphPad Software, La Jolla, CA, USA) was used to analyze the data. One-way analysis of variance was performed to determine statistically significant differences, and then Tukey's post hoc test was performed. A p-value of <0.05 was considered statistically significant.

Expression of miR-4516 was suppressed by TGF-β and TNF-α in RPE cells
To investigate the endogenous expression profile of miR-4516, we first examined the expression of miR-4516 and other well-known microRNAs in RPE cells by qPCR. As shown in Fig  1A, miR-4516 expression is significantly lower than the expression of other well-known micro-RNAs (miR-125, miR-222 and miR-100). Many inflammatory cytokines, such as TGF-β and TNF-α, act as potent inducers of the EMT in RPE cells, which is implicated in the pathogenesis of PVR [1,15,16]. Therefore, we next determined whether these cytokines affected the miR-4516 expression in the RPE cells. By q-RT-PCR, we demonstrated that TGF-β2 (10 ng/mL) and TNF-α (10 ng/mL) significantly inhibited miR-4516 expression in RPE cells ( Fig 1B). TGF-β is known to have three isoforms: TGF-β1, TGF-β2, and TGF-β3, among which TGF-β2 is the major isoform. Furthermore, previous study results suggest that TGF-β2 activates other growth factors that contribute to PVR [1,17]. Therefore, this study used TGF-β2 (10 ng/mL) in the subsequent experiments.

TGF-β2-induced transdifferentiation of RPE cells was inhibited by miR-4516 delivery
RT-qPCR revealed that the level of miR-4516 in the cells was 12,000 times higher than that in ARPE-19 cells transfected with NC at the 24-hour time point. The expression levels decreased gradually over time but remained 4000 times higher than that of cells transfected with NC, even at the 72-hour time point (Fig 2A). We also examined whether miR-4516 is involved in TGF-β2-induced RPE transdifferentiation. As shown in Fig 2B, after treatment of TGF-β2 for 24 hours, fibroblastic transition of the ARPE-19 cells was induced. However, RPE transdifferentiation was significantly suppressed in miR-4516-transfected RPE cells. Cells incubated in the absence of TGF-β2 and transfected with miR-4516 displayed an epithelial morphology.

Biomarker expression of EMT in TGF-β2-treated ARPE-19 cells through miR-4516 delivery
Western blotting was used to examine the protein expression of EMT, including ZO-1, α-SMA, and E-cadherin (epithelial and mesenchymal markers, respectively). As shown in Fig 3A  and 3B, quantitative immunoblot analyses revealed that, compared to control group, TGF-β2-treated ARPE-19 cells had significantly higher levels of α-SMA and significantly lower levels of ZO-1 and E-cadherin, representing epithelial proteins. However, compared to TGF-β2 treated cells or cells transfected with NC, cells transfected with miR-4516 after TGF-β2 treatment exhibited reduced α-SMA expression and increased expression of ZO-1 and E-cadherin. Immunocytochemistry analysis-specifically immunofluorescence-was used to examine the  effects of transfection of ARPE-19 cells with NC or the miR-4516 mimic on the TGF-β2-induced expression of ZO-1 and vimentin (another mesenchymal marker). Consistent with the Western blotting results, ZO-1 and vimentin expression was lower and higher in TGF-β2 treated cells, respectively, than in the control cells. In contrast, in TGF-β2-treated ARPE-19 cells, cells transfected with miR-4516 mimic downregulated and upregulated vimentin and ZO-1, respectively (Fig 3C). However, the expression of these EMT-related proteins was not affected in cells treated with miR-4516 inhibitor (S1 and S2 Figs). Moreover, miR-4516 expression was lower than the expression of other well-known microRNAs (miR-125, miR-222, and miR-100) (Fig 1A). This indicated that miR-4516 inhibitor did not affect the expression of EMT-related proteins due to the low expression of endogenous miR-4516 in APRE-19 cells. Taken together, these results demonstrate that miR-4516 transfection suppressed TGF-β2-induced EMT in the ARPE-19 cells.

Migration of TGF-B2-treated ARPE19 is passivated by miR-4516 delivery
TGF-β2-induced EMT in RPE cells is an initiating event in many fibrotic processes, including collagen contraction, cell migration and proliferation, which occur during PVR pathogenesis [14,18]. Therefore, we studied the effects of miR-4516 transfection on cell migration in TGF-β2-treated ARPE-19 cells using a scratch wound healing assay. Results showed that treatment with TGF-β2 alone enhanced wound closure in a time-dependent manner. At 48 hours, miR-4516 transfection had completely suppressed TGF-β2-induced wound closure (Fig 4A). In sum, miR-4516 transfection inhibited wound closure in TGF-β2-treated ARPE-19 cells. Because cell proliferation and migration are essential to wound closure, we used mitomycin-C to suppress cell proliferation. Therefore, the results suggest that miR-4516 transfection inhibited TGF-β2-induced wound closure by suppressing cell migration (Fig 4A). Next, we used a modified Boyden chamber method to quantify cell migration in the ARPE-19 cells. As shown in Fig 4B, compared with that in the NC cells, cell migration (purple color) was significantly promoted and suppressed in the ARPE-19 cells, respectively, under TGF-β2 stimulation and miR-4516 transfection (p<0.001). No significant differences were noted between the vehicle control and NC groups. In addition, cell migration was not affected in ARPE-19 cells treated with miR-4516 inhibitor (S3 Fig). In short, the results indicate that miR-4516 transfection inhibited TGF-β2-induced cell migration in the ARPE-19 cells.

Effects of miR-4516 on EMT are exerted through OTX1
TGF-β transduces EMT through MAPK in RPE cells [20,21]. Consequently, we evaluated whether miR-4516 regulates TGF-β2-induced EMT via MAPK signaling pathway. As shown in Fig 5A and 5B, after TGF-β2 treatment, p-ERK expression was significantly reduced in miR-4516-transfected RPE cells compared with those transfected with NC. These results were confirmed through quantitative analyses of immunoblotting assays, which indicated that miR-4516 significantly affected p-ERK expression. To identify putative regulatory targets of miR-4516, in silico analysis was performed using the web server Target Scan, which revealed that miR-4516 had the complementary binding sequences targeting 3'-UTR of OTX1 ( Fig 5C). Next, luciferase reporter vector containing wild-type (WT) or mutated 3'-UTR of OTX1 was constructed and transfected into RPE cells together with miR-4516 mimics or miR-NC ( Fig  5D). As expected, overexpression of miR-4516 specifically inhibited the luciferase activity in cells transfected with reporter vector containing WT 3'-UTR of OTX1, but not with reporter vector containing mutated 3'-UTR of OTX1 (Fig 5D). Subsequent immunoblotting assay also exhibited that miR-4516 significantly inhibited TGF-β2-induced OTX1 expression (Fig 5E and  5F). Taken together, these results indicate that miR-4516 transfection inhibited TGF-β2-induced EMT and PVR development in RPE cells, at least in part, through suppression of the OTX1 signaling pathway.

PVR is inhibited by miR-4516 overexpression in vitro and in vivo
To determine whether miR-4516 regulates TGF-β2-induced PVR of RPE cells, the effects of miR-4516 transfection on collagen gel contraction in TGF-β2-treated ARPE-19 cells were evaluated using a collagen matrix contraction assay. Specifically, freshly polymerized collagen matrices containing ARPE-19 cells were assessed for collagen gel extraction. By collagen gel contraction assay, the area of matrices significantly shrunk in TGF-β2-treated group compared to that in the untreated group (Fig 6). However, this shrinkage was attenuated by the overexpression of miR-4516 (Fig 6), and the development of PVR was not affected in cells treated with miR-4516 inhibitor (S4 Fig). These findings suggest that overexpression of miR-4516 reduced the effect of collagen gel contraction in the TGF-β2-treated RPE cells.
The in vivo effect of miR-4516 on PVR development was also examined by injecting 0.1 mL of 10 ng/mL TGF-β2 and 0.1 mL of BSS containing approximately 1 × 10 6 ARPE-19 cells transfected with miR-4516 and miR-NC into rabbits' right eyes. A total of four doses were administered to each eye on days 1, 8, 15, and 22. PVR progression was assessed according to the Fastenberg's classification (S1 Table), which were grouped into 6 stages including stage 0 (without evidence of fibrous ingrowth), 1 (intravitreal membrane), 2 (focal traction; localized vascular changes; hyperemia; engorgement; dilation; blood vessel elevation), 3 (localized detachment of medullary ray), 4 (extensive retinal detachment; total medullary ray detachment; peripapillary retinal detachment), and 5 (Total retinal detachment; retinal folds and holes). As shown in Fig 7A and 7B, in the control group, severe retinal detachment (stages 3 and 4 in the Fastenberg classification) was observed in all rabbits 37 days after injection, although the PVR progress of all rabbits diminished (stage 1 in the Fastenberg classification). S2 Table summarizes the disease status of the rabbits (staged using the Fastenberg classification). As shown in Fig 7B, miR-4516 significantly inhibited PVR development. Ultrasound images revealed severe retinal detachment in the eyes of the control rabbits. The rabbits treated with miR-4516 did not develop this condition (Fig 7C). Hematoxylin and eosin staining showed that compared with the eyes treated with miR-4516, the retinal surface of the eyes treated with miR-NC had dense, fibrous vascular membrane tissue and significantly higher inflammatory cell infiltration (Fig 7D). Compared with eyes injected with ARPE-19 cells transfected with miR-NC, these results show that the progression of PVR in eyes injected with ARPE-19 cells transfected with miR-4516 was significantly inhibited, which indicates that miR-4516 has the potential to prevent and treat PVR.

Discussion
In the present study, for the first time, a novel microRNA (miR-4516) was introduced and identified as being involved in the TGF-β2-induced PVR development in vitro and in vivo. Overexpression of miR-4516 reduced the TGF-β2-induced transdifferentiation and migration in RPE cells. TGF-β2-induced expression of p-ERK and EMT-related proteins were inhibited in RPE cells. In particular, a ERK-related protein, OTX1, was down-regulated in miR-4516-overexpressing RPE cells. Based on these findings, we suggest that overexpression of miR-4516 may down-regulate TGF-β2-induced PVR via the OTX1/ERK/EMT signaling pathway (Fig 8).
In EMT, epithelial cells lose their characteristic epithelial morphology and phenotype and obtain mesenchymal morphology and phenotype [39]. Transdifferentiation is characterized by morphological changes such as decreased cell adhesion and increased cell migration and invasion. Phenotypic changes include decreased expression of epithelial markers such as ZO-1 and E-cadherin, and increased expression of mesenchymal markers such as α-SMA, vimentin, and fibronectin [40]. The essential polypeptide ZO-1 is found in tightly bound complexes, while Ecadherin is found in another type of cell-cell adhesion, adhesion binding. The structure of tight junctions and adherens junctions is important in the structure and function of normal epithelial cells [40][41][42]. In mesenchymal markers, α-SMA is an intracellular cytoskeletal contractile protein involved in cell movement. Vimentin is another intracellular cytoskeletal protein that plays a key role in stabilizing cell structure during migration [40]. The present study expanded these findings and further showed that miR-4516, a novel microRNA, reduced TGF-β2-induced transdifferentiation, migration and EMT-related proteins (α-SMA and ZO-1) in RPE cells. These findings imply that miR-4516 may suppress transdifferentiation of RPE cells by regulating epithelial protein and intracellular cytoskeletal contractile protein.
PVR is an eye disease characterized by the formation of a dense fibrosis shrinkage membrane consisting of extracellular matrix (ECM) and various cell types in the vitreous cavity and anterior retinal surface [43]. RPE cells are closely connected pigment cell monolayers and are the most important PVR component [43]. Typically, RPE cells remain stationary and maintain their characteristic morphology and function. The external blood-retinal barrier (BRB) usually prevents fluid from the choroid vessels from entering the retina. In a pathological state called retinal rupture, BRB breaks down and RPE cells are exposed to vitreous bodies containing various growth factors and cytokines. Many growth factors and cytokines, including plateletderived growth factor, FGF, epidermal growth factor, TGF-β, IL-1, IL-6, IL-8, IL-10 and interferon γ, are strong inducers of EMT and are upregulated in the eyes of PVR [1,15,16]. RPE cells activated by growth factors and cytokines subsequently undergo EMT, and the resulting dedifferentiated RPE cells migrate, proliferate, and transform into proliferative migratory spindle cells, resulting in PVR [42][43][44]. These transdifferentiated spindle cells migrate along the subretinal plane and assume a position of the retina by migrating through retinal fracture. In the present study, overexpression of miR-4516 reduced the effect of collagen gel contraction in the TGF-β2-treated RPE cells. In rabbit models, PVR progression was significantly inhibited in the eyes of miR-4516 cells transfected by injection ARPE-19 cells [14]. Meanwhile, RPE cells undergo EMT, resulting in PVR [42,43]. Another rabbit animal model study also noted that TGFβ induced EMT was associated with PVR progression [18]. Thus, we speculated that miR-4516 suppresses TGF-β2-induced progression of PVR due to inhibition of EMT, which further suggests that miR-4516 has the potential to prevent and treat PVR.
TGF-β is known to transmit its signal through two main pathways, including Smad-dependent pathway and non-Smad pathway [45]. Typical TGF-β/Smad signaling transmits signals by binding to two related type I and type II transmembrane receptors, which further phosphorylate the receptor-regulated Smad protein-Smad2 and/or Smad3 [45]. Phosphorylated Smads work Smad4 with the co-mediator, then translocate to the nucleus and mediate gene transcription [45]. Deckers et al. reported that small interfering RNA targeting Smad4 have been reported to inhibit TGFβ induced EMT in cancer cells [46]. In our previous study, overexpression of miR-1285 inhibited the TGF-β/Smad4 signaling, which both reduced the expression of mesenchymal markers and inhibited the EMT of RPE cells [47]. In contrast, the present study found that overexpression of miR-4516 significantly reduced TGF-β2-induced expression of phosphorylated ERK in RPE cells. Studies have also shown that downregulation of ERK signaling pathways inhibits proliferation, migration and collagen I mRNA expression in RPE cells [48]. Inhibition of the ERK signaling pathway reveals a promising new perspective on treatment and prevention of early PVR [49]. Given these views of ERK, we speculate that miR-4516 inhibited TGFβ induced PVR may be via regulation of the ERK signaling pathway. In addition, other non-Smad signals are involved in the TGF-β of EMT induced in different types of cells, including ERK, p38, and PI3K/AKT methods [21,50,51]. In addition, the nonclassical signal p38 and PI3K/AKT pathways crosstalk and integrate with the Smad pathway to adjust to each other [51,52]. To complicate matters, these non-canonical TGF-β signaling and typical Smad signaling can also be mediated through other signaling pathways, such as Notch pathway [45]. In the present study, we did not evaluate the effect of miR-4516 on TGF-βrelated Smad-and PI3K/AKT-dependent pathways in RPE cells. Thus, further investigation is needed to determine whether overexpression of miR-4516 inhibits TGF-β2-induced EMT in RPE cells via Smad-and PI3K/AKT-dependent pathways.
Micro RNA (miRNA), approximately 18 to 24 nucleotides in length, is a small molecule of endogenous non-coding RNA that can act as a post-transcriptional regulator. MiRNAs interact with the 3' untranslated region (3'-UTR) of their target genes to regulate about 30%-50% of protein-coding genes [53]. Huang et al. demonstrated that miR-1285 inhibits the migration and invasion of pancreatic cancer cells and act as tumor suppressor [54]. Overexpression of Mir-1285 significantly inhibits breast cancer cell proliferation [55]. In our previous study, miR-1285 overexpression decreased the Smad4 3'-UTR activity in RPE cells [47]. In the present study, we performed in silico analysis using the web server Target Scan, which revealed that miR-4516 had the complementary binding sequences targeting 3'-UTR of OTX1, SOX4, GRB2 and ETV4, demonstrating that overexpression of miR-4516 significantly decreased 3'-UTR activity of OTX1 in RPE cells. However, overexpression of miR-4516 did not influence TGF-β2-induced expression of SOX4, GRB2, and ETV4 in ARPE cells, although previous studies have reported that SOX4, GRB2, and ETV4 contribute to EMT in cancer cells [56][57][58][59]. In cancer cells, miR-4516 negatively regulates OTX1 [35], and the ability of cell migration and invasion is reduced after down-regulation of OTX1 [60]. When OTX1 expression is silenced, the cell proliferation ability and the expression level of related protein p-ERK are reduced, indicating that the role of OTX1 in cancer cells may depend on the activation of p-ERK [60]. As the integration point of various biochemical signals, ERK is involved in a variety of cellular processes, including differentiation, proliferation, transcriptional regulation, and development [61]. Interestingly, we found that a ERK-related protein, OTX1, can be targeted by miR-4516 in RPE cells, which thereby reduces OTX1 promoter activity. Moreover, decreased levels of the associated molecules N-cadherin and vimentin may explain a decrease in cell metastasis capacity [36,60], as N-cadherin and vimentin are important molecular markers of EMT. In the present study, the EMT-related markers, including α-SMA, ZO-1 and vimentin, were also regulated by miR-4516 in RPE cells. Based on these findings, we suggest that miR-4516 repression of EMT-related PVR may occur in an OTX1/ERK-dependent manner.
The importance of miRNAs in extracellular space has been confirmed by many studies, and exosome-mediated cell exports of miRNAs are the most important features, as well as their uptake and functional consequences in receptor cells [62][63][64][65]. In addition to providing delivery strategies for drugs or RNA therapeutics, exosomal components can also act as biomarkers to aid diagnosis and help determine treatment options and outcomes. Luga et al. [66] observed that exosomes derived from cancer-associated fibroblasts (CAFs) promoted EMT in recipient breast cancer cells via activating autocrine WNT-planar cell polarity signaling. Recently, miR-543-enriched exosomes induced EMT in RPE cells, suggesting PVR [67]. In the present study, miR-4516 can be used as a biomarker for EMT-related PVR advances. Previous study has shown that miR-4516 is a circulating miRNA [68]. The level of miR-4516 is about 5 times lower in CAF-derived exosomes than normal fibroblast-derived exosomes and are associated with EMT in these cells [69]. Based on these findings, we suggest that further investigations should be conducted to evaluate the level of miR-4516 in RPE-derived exosomes during PVR development and to determine whether or not it is a potential biomarker for PVR.

Conclusions
Results of the present study show that miR-4516 acts as a biomarker for PVR progression. Overexpression of miR-4516 suppresses collagen gel contraction, transdifferentiation, migration, and EMT-related proteins in RPE cells, regulated by the OTX1/ERK signaling pathway. Therefore, miR-4516/OTX1/ERK may serve as a novel therapeutic target for miRNA-based therapy in PVR.